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. 2021 Mar 30;118(13):e2022373118.
doi: 10.1073/pnas.2022373118.

Conservation of the HBV RNA element epsilon in nackednaviruses reveals ancient origin of protein-primed reverse transcription

Jürgen Beck  1 , Stefan Seitz  2 , Chris Lauber  3   4 , Michael Nassal  5
Affiliations

Conservation of the HBV RNA element epsilon in nackednaviruses reveals ancient origin of protein-primed reverse transcription

Jürgen Beck et al. Proc Natl Acad Sci U S A. .

Abstract

Hepadnaviruses, with the human hepatitis B virus as prototype, are small, enveloped hepatotropic DNA viruses which replicate by reverse transcription of an RNA intermediate. Replication is initiated by a unique protein-priming mechanism whereby a hydroxy amino acid side chain of the terminal protein (TP) domain of the viral polymerase (P) is extended into a short DNA oligonucleotide, which subsequently serves as primer for first-strand synthesis. A key component in the priming of reverse transcription is the viral RNA element epsilon, which contains the replication origin and serves as a template for DNA primer synthesis. Here, we show that recently discovered non-enveloped fish viruses, termed nackednaviruses [C. Lauber et al., Cell Host Microbe 22, 387-399 (2017)], employ a fundamentally similar replication mechanism despite their huge phylogenetic distance and major differences in genome organization and viral lifestyle. In vitro cross-priming studies revealed that few strategic nucleotide substitutions in epsilon enable site-specific protein priming by heterologous P proteins, demonstrating that epsilon is functionally conserved since the two virus families diverged more than 400 Mya. In addition, other cis elements crucial for the hepadnavirus-typical replication of pregenomic RNA into relaxed circular double-stranded DNA were identified at conserved positions in the nackednavirus genomes. Hence, the replication mode of both hepadnaviruses and nackednaviruses was already established in their Paleozoic common ancestor, making it a truly ancient and evolutionary robust principle of genome replication that is more widespread than previously thought.

Keywords: HBV long-term evolution; HBV replication mechanism; initiation of reverse transcription; paleovirology; protein priming.

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Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.
Identification and characterization of the RNDV replication origin RNDVε. (A) Mapping of RNDVε (red box). RNDV P was expressed in a coupled in vitro transcription/translation (IVT) system from construct P1 or from P2 complemented with the indicated RNDV UTR RNA fragments (wavy lines, RNA transcripts; T7, T7 promoter). Priming assays were performed, and guanylylation of P was assessed by SDS polyacrylamide gel electrophoresis and phosphoimaging. The top band represents the 72 kDa full-length RNDV P, the band below a truncated yet functional RNDV P translation product. Numbers refer to genomic nt positions as previously defined (2). (B) Position of RNDVε in the RNDV genome. The predicted pgRNA start site and the polyA signal are indicated by arrow and diamond, respectively; s1, s2, and s3 denote nackednavirus specific small ORFs (smORFs) of unknown function. (C) The 5′-proximal position of ε on the pgRNA is conserved between RNDV and HBV. pgRNAs are depicted as wavy lines; terminal redundancy (R), polyA tail (pA). HBV contains a second copy of ε in the 3′ redundancy (gray) dispensible for replication (4). (D) Secondary structure of RNDVε predicted by MC-Fold. (E) The RNDV reverse transcription initiation site maps to RNDVε nt C51. RNDV P was expressed from P2, and nucleotidylation of P was assessed using the indicated C to U point mutants of RNDVε plus [α-32P]dGTP or [α-32P]dATP as substrate. (F) (−)DNA primer (red) and structural context of the primer template of RNDV, DHBV, and HBV. The Tyr residue (Y) of TP, covalently attached to the initiating G residue, is indicated, and the templating C is highlighted in orange. The length of the RNDV primer has not been determined, but the limited complementarity to the putative primer transfer site suggests it does not exceed two nt. HBV possesses a potential alternative initiation site (dashed line to bracketed T).
Fig. 2.
Fig. 2.
Comparison of nackednaviral and hepadnaviral ε elements. (A) Alignment of nackednaviral and hepadnaviral ε sequences. Colored letters indicate conserved sequence elements in lower stem (red), initiation region (orange), and apical region (blue). Base-paired lower stem regions are shaded in blue, and the variably base-paired tip of the lower stem is in light blue. A black arrowhead denotes the intiation site. The smORF1 start codons of nackednaviruses and the core start codons of HBV and DHBV are underlined. Internal numbers denote nt not depicted in the figure. Italic numbers refer to genomic nt positions (2). African cichlid nackednavirus (ACNDV), European eel nackednavirus (EENDV), Western mosquitofish nackednavirus (WMNDV), Lucania parva killifish nackednavirus-1 and -2 (KNDV-Lp1, KNDV-Lp2), Astatotilapia nackednavirus (ANDV), sockeye salmon nackednavirus (SSNDV), baby whale nackednavirus-1 (BWNDV-1), and stickleback nackednavirus (SNDV). (B) Consensus ε structures of nackednaviruses (Left), avihepadnaviruses (Middle), and orthohepadnaviruses (Right). Dashed lines between nt indicate nonconserved, facultative bp, and gray lines indicate potential noncanonical bp.
Fig. 3.
Fig. 3.
Functional adaptation of nackedna- and hepadnaviral ε elements to heterologous P proteins. Few strategic nt exchanges in ε enable cross-species P protein priming. (A) RNDV/DHBV chimeric ε variants. Substituted nt are encircled. RNDV-specific nt are in blue, and DHBV-specific nt are in orange. Note that the RNDVε variant R4 and the DHBVε variant D4 contain reciprocal substitutions at homologous positions. (B) RNDV and DHBV P guanylylation activities of RNDVε variants R1 to R6. P proteins were expressed from ε-deficient templates, and priming assays were performed in the presence of the indicated ε variants (1 µM) as described in Fig. 1A. (C) Guanylylation activities of DHBV P adapted RNDVε variant R4 (Left) and RNDV P adapted DHBVε variant D4 (Right) at different ε concentrations. Maximal priming activity of the cognate ε was set to 100%. Only the upper/full-length band of RNDV P is shown. (D) Chimeric ε variants for adaptation of HBVε to DHBV and RNDV P. DHBV-specific nt are in orange, RNDV-specific nt are in blue, and nt identical between DHBV and RNDV are in red. Note that HBVε wt* contains structure destabilizing mutations (gray) essential for HBV P in vitro priming activity which do not affect P specificity. (E) DHBV, RNDV, and HBV P guanylylation activities of HBVε variants H1 to H5 (performed as described in B). (F) Compatibility of ε determinants for priming of reverse transcription between nackedna- and hepadnaviruses. Cross-functionality of heterologous pairs of P and ε is indicated by identical background color of circular icons. RNDV (R), DHBV (D), HBV (H).
Fig. 4.
Fig. 4.
Model of nackednaviral replication. (A) Alignment of replication relevant cis elements of nackednaviruses. Italics indicate genomic positions. Internal numbers denote nt not depicted in the figure. The conserved consensus initiator element (30) (Inr, boxed in gold) harbors the predicted pgRNA initiation site (+1) located at a canonical distance of 31 nt to the conserved TATA box of the pgRNA promoter. Boxed nt indicate mapped polyA attachment sites. Template switches are indicated by encircled numbers. The GT primer template in ε and the primer transfer site are shaded in gray. Magenta arrowheads mark RNase H cleavage sites for (+) strand primer generation. (B) Model for hepadnavirus-like reverse transcription of nackednaviral pgRNA into rcDNA. A TP-linked GT primer is copied from ε (white box) and transferred to a conserved AC 3′ element (red) in the terminal redundancy (R). The entire pgRNA is copied into (−)DNA by primer elongation, and the pgRNA is degraded (dashed gray line) by P’s RH activity, leaving a short RNA oligo (red line), which serves as (+)DNA primer upon transfer to complementary DR2. A third template switch from the 5′ to the 3′ end of (−)DNA fostered by the short terminal redundancy (r) of five to six nt (detailed in shaded inset, hepadnaviruses eight to nine nt) accomplishes circularization, and (+)DNA elongation generates HBV-like rcDNA. Note that the RT domain of P linked to the (−)DNA is engaged in DNA synthesis and associated with the 3′ end of nascent DNA, which is not depicted in the figure.
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